Project Summary Engineering natural product biosynthesis in bacteria represents a promising strategy to produce these high-value and pharmaceutically important compounds. However, to achieve economically viable production is quite difficult and challenging. To address this challenge, establishing artificial logic and dynamic control functions in cells by mimicking natural regulations in biological systems has been developed and becomes a powerful approach to enable superior microbial production. This approach renders great biological robustness to the cells and allows them to autonomously adjust their metabolic states and activities by sensing the cellular and environmental changes. However, compared with natural regulations that exert simultaneous and orthogonal up- and down- regulation on various gene targets, the current dynamic control techniques mainly relies on simple gene circuits to perform mono-function, either up- or down-regulation, on a limited set of genes, which are still rudimentary and less sophisticated. To bridge these gaps, development of new genetic tools and their use in novel control strategies are highly desired. Recently, the PI?s lab has achieved the biosynthesis of natural products with defined pharmaceutical properties and therapeutic effects such as 4-hydroxycoumarin and 5-hydroxytryptophan in E. coli through synthetic biology, metabolic engineering and protein engineering approaches and developed antisense RNA tools for intervening cellular metabolism to enhance the biosynthesis of plant polyketides, such as flavanone naringenin. In this MIRA proposal, we aim at exploring a new and general strategy for developing more sophisticated dynamic control network to greatly enhance natural product biosynthesis in bacteria. The dynamic control network is expected to have the capability of implementing autonomous and intelligent up- regulation and down-regulation functions in response to cellular and environmental changes and maintain the cells at optimal production states throughout all growth stages to achieve maximal production efficiency. Our recent progress on the biosynthesis of 4-hydroxycoumarin, 5-hydroxytryptophan and the development of antisense RNAs tools will serve as the groundwork for us to understand how to efficiently establish and implement the dynamic control network and decision-making strategies in cells for real-world applications. Specifically, four coherent projects with distinct research activities will be pursued, which include: 1) developing salicylic acid, p-coumaric acid and tryptophan responsive promoters for up-regulation of gene expression; 2) expanding antisense RNA tools for controllable down-regulation of gene expression; 3) developing and characterizing promoter and antisense RNA based dynamic control network; 4) applying dynamic control network to improve biosynthesis of 4-hydroxycoumarin, flavanone naringenin and 5-hydroxytryptophan in E. coli.